<p> mRNA Isolation using Oligotex Spin-Column</p><p>Materials and equipment necessary for this protocol:</p><p>Oligotex Kit (Qiagen) Contained in this kit: Oligotex beads, OEB Buffer, OBB Buffer, OW2 Buffer, RNase-Free water, spin-columns, collection tubes.</p><p>10 mM Tris-Cl pH 7.5 (RNase-Free)</p><p>Heat blocks set to 37C and 70C (avoid using water baths – it is much easier to avoid RNase contamination using dry heat blocks), microcentrifuge, shaker/rocker (not necessary but helpful).</p><p>Protocol: 1. Heat Oligotex suspension to 37C. Vortex and leave at room temp. 2. Heat OEB Buffer to 70C. Leave there until further use. 3. Heat OBB buffer to 37C to re-dissolve any precipitated material. Allow it to cool to room temp. 4. Dilute total RNA with water to appropriate volume depending on RNA content.</p><p>Total RNA Add RNAse Free water to: < 0.25 mg 250 l 0.25 - 0.50 mg 500 l 0.50 - 0.75 mg 500 l 0.75- 1.00 mg 500 l</p><p>5. Add equal volume of OBB Buffer and mix. 6. Add appropriate volume of Oligotex suspension depending on RNA content and mix well.</p><p>Total RNA Oligotex Suspension < 0.25 mg 15 l 0.25 - 0.50 mg 30 l 0.50 - 0.75 mg 45 l 0.75- 1.00 mg 55 l</p><p>7. Incubate at 70C for 5 min. (and put it on ice for several min.) 8. Allow to sit at room temp for 30 min. Invert tubes frequently to ensure better binding. You can put tubes in a test tube rack (on it’s side) on a shaker/rocker set to rotate at a very slow setting. Set it very slow so the solution just moves back and forth from one end of the tube to the other gently. (Use agitator to invert the samples.) 9. Pellet the Oligotex:mRNA complex for 2 min at max speed at room temp. 10. Remove supernatant and do not disturb the pellet. It is OK to leave 25 to 50 l so that there is no loss of pellet. (Save supernatant on ice until protocol is completed). 11. Re-suspend pellet in 400 l of OW2 Buffer. Add suspension to spin-column. 12. Spin at maximum for 1 min. 13. Remove flow-through from lower portion. Apply 400 l new OW2 Buffer to column. 14. Spin at maximum for 1 min. 15. Transfer spin-column to new RNase-free tube. Place in the 70C heat block alongside of OEB Buffer. 16. Not allowing solution to cool, add 20 l OEB Buffer to column and completely re- suspend the resin by pipetting. Leave tubes (with columns) at 70C until all samples are completely re-suspended. 17. Spin at maximum for 1 min. 18. Recover the elution volume (20 l) from lower portion and USE THAT SAME VOLUME to repeat step 16. This is IMPORTANT to maintain the final volume at 20 l. This will ensure that the mRNA concentration will be high enough for the subsequent probe labeling reaction. 19. Spin at maximum for 2 min. 20. Save the elution volume (20 l) on ice for quantitation. It may be necessary to re-adjust the volume to 20 l with OEB Buffer (some of the volume may have been lost during subsequent steps due to evaporation, etc). 21. Quantify samples by obtaining absorbance at 260 and 280 nm. Dilute 1 µl of total RNA sample 1:100 using 10 mM Tris-Cl pH 7.5 (1 µl RNA + 99 µl buffer) and mix well by pipetting. Obtain absorbance readings using a clean microcuvette using the diluted sample. Discard diluted samples once absorbance has been obtained. Use the accompanying RNA quantitation template to calculate RNA yields and purity. 22. Once concentrations are determined, one can set up and begin the cDNA synthesis reaction for generation of microarray probes or store mRNA samples at -80ºC.</p>